Development of Agrobiological Resources, Faculty of Agriculture, Meijo University Nagoya, Japan.

Abstract

Gene targeting (GT) refers to the designed modification of genomic sequence(s) through homologous recombination (HR). GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ) occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS) is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice-a major crop worldwide-reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology based on PNS are discussed.

Schematic diagram of various gene modifications by PNS-mediated GT. (A) The brown box indicates the gene to be targeted on a genome sequence shown as black lines. The brown arrow represents the promoter of the gene. (B) PNS vector for GT. The green arrows are the negative markers; the red arrow is the positive marker. The pink box is the transcriptional stop sequence of En/Spm. The gray arrows are loxP sequences. Double-headed arrows under the vector indicate the homology regions for HR. The blue line is T-DNA sequence. (C) HR process for GT between the target gene and PNS vector. The thick lines of black and blue indicate newly synthesized DNA sequences in genome and T-DNA, respectively. (D) Gene knock-out of the target gene by insertion of a positive marker with En/Spm, which can be removed via subsequent Cre-loxP recombination caused by introduced Cre gene (yellow arrow). (E) Reactivation of knock-out gene in (D) by Cre-loxP recombination. (F) Nucleotide(s) substitution (red lines), insertion, and deletion in the target gene can be induced by designing a homology arm in the PNS vector in (B) and subsequent positive marker elimination by Cre-loxP recombination in (D). (G) Gene knock-in modification where the endogenous promoter sequence is connected to the GUS coding sequence (indicated as a blue box with En/Spm). (H) Gene knock-in modification where the mOrange coding sequence, indicated as an orange box, is connected precisely to the stop codon of the target gene; both endogenous promoter activity and protein localization of the target gene are detectable. (I–K) Diagrams of segregated plants from knock-in T0 into homozygote (I), heterozygote (J), and wild type (K). GUS expression image as blue leaves is shown in (I,J). Dwarf phenotype in (I) is a reflection of the disrupted target gene.

Strategy for the introduction of point mutations into the ALS locus via GT and subsequent marker excision from the GT locus using the piggyBac transposon. (A) Schematic diagram of GT at the ALS locus. The top line indicates the genomic structure of the wild-type ALS gene region. The bottom line shows the T-DNA region of the GT vector carrying DT-A as a negative selection marker and a 6.4-kb fragment containing an ALS coding region (gray box) with W548L and S627I mutations (white lines) and silent mutations (TTAA site added 301-bp upstream of W548L; GCTGAC to GAATTC) for the insertion of piggyBac transposon harboring hpt gene as a positive selection marker. The W548 L and S627I mutations create novel Mfe I restriction sites (M). LB, left border; RB, right border. (B) Strategy for precise marker excision from the GT locus using piggyBac transposon. The top line reveals the structure of the modified ALS locus resulting from HR between the GT vector and wild-type locus. The bottom line represents the ALS locus modified by GT and subsequent precise marker excision via piggyBac transposition. The primer sets used for PCR that identify transgenic calli in which a GT event occurred at the ALS locus are shown as black arrows. White arrows indicate the primer sets used for CAPS analysis to evaluate the frequency of marker excision via piggyBac transposition. The numbers on each arrow reveal the length of the PCR fragments.